Sequence analysis indicated that dengue type 4 viral genome contains 384 nucleotides in the 3' non-coding region (NCR). The last 82 nucleotides are thought to assume a secondary structure probably required for initiation of RNA replication. Preceding this structure are sets of conserved sequences, designated CS-1 and CS-2, that are also found in other mosquito-borne flaviviruses. We have engineered a series of cDNA constructs containing deletions ranging from 29 to 201 nucleotides in length in the 3' NCR. Mutant RNA transcripts made from these DNA constructs were tested for infectivity by transfecting permissive tissue culture cells. The last 113 nucleotides including the secondary structure at the 3' end and the preceding CS-1 sequence apparently are essential for infectivity of dengue virus. In contrast, viable dengue virus was recovered from mutant DNA constructs containing a deletion in one or the other of the two CS-2 sequences. Many viable deletion mutants constructed in this manner were stable and produced plaques of reduced size on infected C6/35 mosquito cells compared to wild type virus. This suggests that these mutants are restricted for their replication. These mutants are now being characterized further for their replicative capacity and other properties in cultured cells as well as in infected animals. Dengue virus mutants that show reduced virulence for animals will be evaluated subsequently in humans for evidence of attenuation and immunogenicity.